Synthesis Of 2'-deoxyuridine By Combined Chemical-Biologic Method

2'-deoxy-5-fluorouridine is a 5-fluorinated deoxyuridine derivative,now it is still widely used in the clinical treatment of tumors.Due to the complex and strict conditions of chemical synthesis process,it is significant if we could find a greener way.Here we used a combined chemical-biologic method,trying to solve this problem.In this article,1-chloro-2-deoxy-3,5-O-di-(4-chlorobenzoyl)-D-ribo-furanose was used as raw material to gain the key intermediate 2-deoxy-a-D-ribose-1-phosphate by crystallization-induced asymmetric transformation.It was used with 5-Fu as substrates in the next biotransformation step to synthesize 2'-deoxy-5-fluorouridine.One strain with high nucleoside phosphorylase activity was screened as the enzyme source,and processes were investigated to gain high conversion rate.The results showed that,the yield of 2-deoxy-a-D-ribosyl-1-phosphate bis(cyclohexylamine)could reach up to 85.84%,its purity was 95.75%according to HPLC analysis,and the structure was consistent with literature according to 1H NMR Spectroscopy and IR.Conditions of enzyme induction and conversion were optimize to improve substrate conversion efficiency.The optimum induction condition for nucleoside phosphorylase of Bacillus cereus s2308 was inducing by 15 mmol/L cytidine for 27 h,and the activity of enzyme was significantly improved,and the optimum condition for conversion we got was 35 mmol/L 2-deoxy-a-D-ribosyl-1-phosphate bis(cyclohexylamine),105 mmol/L 5-Fu dissolved in potassium phosphate buffer(30 mM,pH 8.0),adding 12%(m/v)Bacillus cereus s2308 wet whole cell,55 ? for 3 h,the conversion rate reached up to 79.36%,and the concentration of the product was 27.77 mmol/L.Finally,the immobilization methods of cells were preliminarily studied for the purpose of enzyme reuse.In this study,we found that whether we use agarose,carrageenan or other embedding material,the immobilized cells showed low activity,While cells treated with permeabilizing and crosslinking showed quite preferable result.It showed better activity than free cell when used for the first time,while its optimum reaction pH value and temperature showed no distinct differences from free cell,and it could be reused for 5 times before the enzyme activity decreased to 35.40%of initial activity,comparing to that just 10%of free cell for the second use.This combined chemical-biologic method of nucleoside synthesis can effectively solve the problems such as the lack of deoxyribose donor source,low conversion rate,product poor stereoselectivity,and other issues,and it could also reduce the use of a variety of organic reagents,which shows positive significance for scientific research,industrial applications,and for environmental protection too.This method can also be extended to other nucleoside drugs such as Cladribine,and could provide a new way for green synthesis of such compounds.